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Received 23 Oct 2013 | Accepted 22 Apr 2014 | Published 23 May 2014

Shengyi Liu1,*, Yumei Liu2,*, Xinhua Yang3,*, Chaobo Tong1,*, David Edwards4,*, Isobel A.P. Parkin5,*, Meixia Zhao1,6, Jianxin Ma6, Jingyin Yu1, Shunmou Huang1, Xiyin Wang7,8, Junyi Wang3, Kun Lu9, Zhiyuan Fang2, Ian Bancroft10,Tae-Jin Yang11, Qiong Hu1, Xinfa Wang1, Zhen Yue3, Haojie Li12, Linfeng Yang3, Jian Wu2, Qing Zhou3, Wanxin Wang2, Graham J. King13, J. Chris Pires14, Changxin Lu3, Zhangyan Wu3, Perumal Sampath11, Zhuo Wang3, Hui Guo7, Shengkai Pan3, Limei Yang2, Jiumeng Min3, Dong Zhang7, Dianchuan Jin8, Wanshun Li3, Harry Belcram15, Jinxing Tu16, Mei Guan17, Cunkou Qi18, Dezhi Du19, Jiana Li9, Liangcai Jiang12, Jacqueline Batley20, Andrew G. Sharpe21, Beom-Seok Park22, Pradeep Ruperao4, Feng Cheng2, Nomar Espinosa Waminal11,23, Yin Huang3, Caihua Dong1, Li Wang8, Jingping Li7, Zhiyong Hu1, Mu Zhuang2, Yi Huang1, Junyan Huang1, Jiaqin Shi1, Desheng Mei1, Jing Liu1, Tae-Ho Lee7, Jinpeng Wang8, Huizhe Jin7, Zaiyun Li16, Xun Li17, Jiefu Zhang18, Lu Xiao19, Yongming Zhou16, Zhongsong Liu17, Xuequn Liu24, Rui Qin24, Xu Tang7, Wenbin Liu3, Yupeng Wang7, Yangyong Zhang2, Jonghoon Lee11, Hyun Hee Kim23, France Denoeud25,26, Xun Xu3, Xinming Liang3, Wei Hua1, Xiaowu Wang2, Jun Wang3,27,28,29, Boulos Chalhoub15 & Andrew H. Paterson7

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplication of transposable elements, differential gene co-retention for specic pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

1 The Key Laboratory of Biology and Genetic Improvement of Oil Crops, The Ministry of Agriculture of PRC, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China. 2 The Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, The Ministry of Agriculture, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 10081, China. 3 Beijing Genome Institute-Shenzhen, Shenzhen 518083, China. 4 Australian Centre for Plant Functional Genomics, School of Agriculture and Food Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. 5 Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada S7N OX2. 6 Department of Agronomy, Purdue University, WSLR Building B018, West Lafayette, Indiana 47907, USA. 7 Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia 30605, USA. 8 Center for Genomics and Computational Biology, School of Life Sciences, and School of Sciences, Hebei United University, Tangshan 063000, China. 9 College of Agronomy and Biotechnology, Southwest University, BeiBei District, Chongqing 400715, China. 10 Centre for Novel Agricultural Products (CNAP), Department of Biology, University of York, Wentworth Way, Heslington, York YO10 5DD, UK. 11 Department of Plant Sciences, Plant Genomics and Breeding Institute and Research Institute for Agriculture and Life Sciences, College of Agriculture & Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. 12 Sichuan Academy of Agricultural Sciences, Chengdu 610066, China. 13 Southern Cross Plant Science, Southern Cross University, Lismore, New South Wales 2480, Australia. 14 Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211-7310, USA. 15 Organization and Evolution of Plant Genomes, Unit de Recherche en Gnomique Vgtale, Unit Mixte de Recherche 1165 (Institut National de Recherche Agronomique, Centre National de la Recherche Scientique, Universit Evry Val dEssonne), Evry 91057, France. 16 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. 17 College of Agronomy, Hunan Agricultural University, Changsha 410128, China. 18 Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. 19 Qinghai Academy of Agriculture and Forestry Sciences, National Key Laboratory Breeding Base for Innovation and Utilization of Plateau Crop Germplasm, Xining 810016, China. 20 Australian Research Council Centre of Excellence for Integrative Legume Research, University of Queensland, Brisbane, Queensland 4072, Australia. 21 National Research Council Canada, Saskatoon, Saskatchewan, Canada S7N 0W9. 22 The Agricultural Genome Center, National Academy of Agricultural Science, RDA, 126 Suin-Ro, Suwon 441-707, Republic of Korea. 23 Department of Life Science, Plant Biotechnology Institute, Sahmyook University, Seoul 139-742, Republic of Korea. 24 School of Life Sciences, South-Central University for Nationality, Wuhan 430074, China. 25 Commissariat lEnergie Atomique (CEA), Genoscope, Institut de Gnomique, BP5706 Evry 91057, France. 26 Centre National de Recherche Scientique (CNRS), Universit dEvry, UMR 8030, CP5706, Evry 91057, France. 27 Department of Biology, University of Copenhagen, Ole Maales Vej 5, 2200, Copenhagen, Denmark. 28 King Abdulaziz University, Jeddah, 21589, Saudi Arabia. 29 Department of Medicine and State Key Laboratory of Pharmaceutical Biotechnology, University of Hong Kong, 21 Sassoon Road, Hong Kong. * These are joint rst authors. Correspondence and requests for materials should be addressed to S.L. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 5:3930 | DOI: 10.1038/ncomms4930 | http://www.nature.com/naturecommunications

DOI: 10.1038/ncomms4930 OPEN

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4930

Brassica oleracea comprises many important vegetable crops including cauliower, broccoli, cabbages, Brussels sprouts, kohlrabi and kales. The species demonstrates extreme

morphological diversity and crop forms, with various members grown for their leaves, owers and stems. About 76 million tons of Brassica vegetables were produced in 2010, with a value of14.85 billion dollars (http://faostat.fao.org/

Web End =http://faostat.fao.org/). Most B. oleracea crops are high in protein1 and carotenoids2, and contain diverse glucosinolates (GSLs) that function as unique phytochemicals for plant defence against fungal and bacterial pathogens3 and on consumption have been shown to have potent anticancer properties46.B. oleracea is a member of the family Brassicaceae (B338 genera and 3,709 species)7 and one of three diploid Brassica species in the classical triangle of U8 that also includes diploidsB. rapa (AA) and B. nigra (BB) and allotetraploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC). These allotetraploid species are important oilseed crops, accounting for 12% of world edible oil production (http://faostat.fao.org/

Web End =http://faostat.fao.org/). As the origin and relationship between these species is clear, the timing and nature of the evolutionary events associated with Brassica divergence and speciation can be revealed by inter-specic genome comparison. Each of the Brassica genomes retains evidence of recursive whole-genome duplication (WGD) events9,10 (Supplementary Fig. 1) and have undergone a Brassiceae-lineage-specic whole-genome triplication (WGT)11,12 since their divergence from the Arabidopsis lineage. These events were followed by diploidization that involved substantial genome reshufing and gene losses1115. Because of this, Brassica species are a model for the study of polyploid genome evolution (Supplementary Fig. 2), mechanisms of duplicated gene loss,

neo- and sub-functionalization, and associated impact on morphological diversity and species differentiation.

We report a draft genome sequence of B. oleracea and its comprehensive genomic comparison with the genome of sister species B. rapa, which diverged from a common ancestor B4

MYA. These data provide insights into the dynamics of Brassica genome evolution and divergence, and serve as important resources for Brassica vegetable and oilseed crop breeding. Furthermore, this genome will support studies of the large range of morphological variation found within B. oleracea, which includes sexually compatible crops such as cabbages, cauliower and broccoli that are important for their economic, nutritional and potent anticancer value.

ResultsB. oleracea genome assembly and annotation. Complementing the sequencing of the smaller B. rapa genome11, a draft genome assembly of B. oleracea var. capitata line 0212 was produced by interleaving Illumina, Roche 454 and Sanger sequence data. This assembly represents 85% of the estimated 630 Mb genome, and includes 498% of the gene space (Supplementary Methods,

Supplementary Tables 13, 7 and 8 and Supplementary Fig. 3). The assembly was anchored to a new genetic map16 to produce nine pseudo-chromosomes that account for 72% of the assembly, and validated by comparison with a B. oleracea physical map17, a high-density B. napus genetic map18 and complete BAC sequences (Supplementary Figs 49 and Supplementary Tables 4 and 5). For comparative analyses, identical genome annotation pipelines were used for annotation of protein-coding genes and transposable elements (TEs) for B. oleracea and B. rapa.

Table 1 | Summary of genome assembly and annotation of B. oleracea.

B. oleracea genome assembly

N90 N50 Longest Total size Contig size (bp) 3,527 26,828 199,461 502,114,421

Contig number 22,669 5,425

Total number(42 kb): 27,351Scaffold size (bp) 258,906 1,457,055 8,788,225 539,907,250

Scaffold number 388 224 Anchored to chr. 72%Total number(42 kb): 1,809

B. oleracea genome annotationB. oleraceaIn the assembly In WG short reads* Size (bp) Copy numberw % assemblyz

Retrotransposon 105,755,173 108,948 22.13 23.60 DNA transposon 79,675,583 170,500 16.67 12.71 Total 185,430,756 279,448 38.80 36.31

Gene models Gene space coveredy Annotated Supported by ESTs Protein-coding genes 45,758 98% 91.6% 99.0%

Average transcript length Average coding length No. of average exons No. of alternative splicing variants1,762 bp 1,037 bp 4.6 30,932

Non-coding RNA miRNA tRNA rRNA snRNA Copy number 336 1,425 553 1,442

Average length (bp) 119 75 166 110

*WG, whole genome, 20 coverage reads were randomly sampled from all the genomic short reads libraries.

wThe copy number of TEs was from the RepeatMasker results.

zThe ungapped regions were used to detect the percentage of TEs in the assembly. TE sizes are from the ungapped regions of B. oleracea 477,847,347 bp. yEstimated by public Brassica ESTs and RNA-seq data.

2 NATURE COMMUNICATIONS | 5:3930 | DOI: 10.1038/ncomms4930 | http://www.nature.com/naturecommunications

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A total of 45,758 protein-coding genes were predicted, with a mean transcript length of 1,761 bp, a mean coding length of 1,037 bp, and a mean of 4.55 exons per gene (Table 1, Supplementary Methods, Supplementary Table 6 and Supplementary Fig. 10), similar to A. thaliana19 and B. rapa11. Publicly available ESTs, together with RNA sequencing (RNA-seq) data generated in this study, support 94% of predicted gene models (Supplementary Tables 7 and 8), and 91.6% of predicted genes have a match in at least one public protein database (Supplementary Tables 9 and 10, and Supplementary Fig. 11). Of the 45,758 predicted genes, 13,032 produce alternative splicing (AS) variants with intron retention and exon skipping (Supplementary Table 11). Genome annotation also predicted 3,756 non-coding RNAs (miRNA, tRNA, rRNA and snRNA) (Supplementary Table 12).

A combination of structure-based analyses and homology-based comparisons resulted in the identication of 13,382 TEs with clearly identied terminal boundaries, including 5,107 retrotransposons and 8,275 DNA transposons (Supplementary Methods, Supplementary Fig. 12 and Supplementary Table 13). These elements together with numerous truncated elements or TE remnants make up 38.80% of the assembled portion of theB. oleracea genome, whereas TEs account for only 21.47% of theB. rapa genome assembly. Copia (11.64%) and gypsy (7.84%) retroelements are the major constituents of the repetitive fraction, and are unevenly distributed across each chromosome, with retrotransposons predominantly found in pericentromeric or heterochromatic regions (Supplementary Fig. 13) in B. oleracea.

Tentative physical positions of some of the centromeres were determined based on homologue and phylogenetic analysis of the centromere-specic 76 bp tandem repeats CentBo-1 and CentBo-2 and copia-type retrotransposon (CentCRBo) (Supplementary Table 14 and Supplementary Figs 1417). The distribution of 45S and 5S rDNA sequences were also visualized by uorescent in situ hybridization (Supplementary Figs 18 and 19), leading to a predicted karyotype ideogram for B. oleracea (Supplementary Fig. 20). An extra-centromeric locus with colocalized centromeric satellite repeat CentBo-1 and the centromeric retrotransposon CRBo-1 was observed on the long arm of chromosome 6 (Supplementary Figs 1820). A comprehensive database for the genome information is accessible at http://www.ocri-genomics.org/bolbase/index.html

Web End =http://www.ocri-genomic http://www.ocri-genomics.org/bolbase/index.html

Web End =s.org/bolbase/index.html .

Conserved syntenic blocks and genome rearrangement after WGT. The relatively complete triplicated regions in B. oleracea and B. rapa were constructed and they relate to the 24 ancestral crucifer blocks (AX) in A. thaliana20. Further the triplicated blocks resulting from WGT in the two Brassica species were partitioned into three subgenomes: LF (Least-fractionated), MF1 (Medium-fractionated) and MF2 (Most-fractionated)11 (Fig. 1a, Supplementary Methods, Supplementary Tables 15 and 16, and Supplementary Figs 2126). These syntenic blocks occupy the majority of the genome assemblies of A. thaliana (19,628 genes,72.24% of 27,169 genes), B. oleracea (26,485 genes, 57.88%) andB. rapa (26,698 genes, 64.84%), and provide a foundation for

125 250 375

C01 C02 C03 C04 C05 C06 C07 C08 C09

Chr1 Chr2 Chr3 Chr4 Chr5

A01 A02 A03 A04 A05 A06 A07 A08 A09 A10

B. oleracea

B. rapa

A. thaliana

b c

100

orthologous genes retained

-WGD (~35 MYA)

WGT (~15.9 MYA)

~20 MYA

~10 MYA

Bol_LF Bra_LF

Bol_MF1 Bra_MF1

Bol_MF2 Bra_MF2

A. lyrata ~207 Mb, n=6

Proportion of

~8 MYA

~4.6 MYA

~630 Mb, n=9B. nigra ~630 Mb, n =8

A. thaliana

~135 Mb, n=5

Arabidopsis lineage

Brassica lineage

B. rapa

~485 Mb, n=10

B. oleracea

chr1 chr2 chr3 chr4 chr5

Figure 1 | Genomic structure and gene retention rates in syntenic regions of B. oleracea and B. rapa. (a) Segmental colinearity of the genomesof B. oleracea, B. rapa and A. thaliana. Syntenic blocks are dened and labelled from A to X (coloured) previously reported in A. thaliana20. (b) Time estimate of WGD and subsequent two Brassica species divergence. (c) Pattern of retention/loss of orthologous genes on each set of three subgenomic (LF, MF1 and MF2) blocks of B. oleracea and B. rapa corresponding to A. thaliana A to X blocks. The x axis denotes the physical position of each A. thaliana gene locus. The y axis denotes the proportion of orthologous genes retained in the B. oleracea and B. rapa subgenomic blocks around each A. thaliana gene, where 500 genes anking each side of a certain gene locus were analysed, giving a total window size of 1,001 genes.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4930

comparative analyses of chromosomal rearrangement, gene loss and divergence of retained paralogues after WGT. Massive gene loss occurred in an asymmetrical and reciprocal fashion in the three subgenomes of each species and was largely completed before the B. oleraceaB. rapa divergence (Fig. 1c, Supplementary Tables 1719 and Supplementary Figs 2527). The timing of this evolutionary process was supported by the estimated timing of WGT B15.9 million years ago (MYA), and species divergence B4.6 MYA, based on synonymous substitution (Ks) rates of genes located in the blocks (Fig. 1b and Supplementary Table 20). Gene loss occurred mainly through small deletions that may be caused by illegitimate recombination21,22 (Supplementary Fig. 27), consistent with observations in other plant genomes.

Abundant genome rearrangement following WGT and subsequent Brassica species divergence resulted in complex mosaics of triplicated ancestral genomic blocks in the A and C genomes (Fig. 1a and Supplementary Fig. 28). At least 19 major, and numerous ne-scale, chromosome rearrangements occurred, which differentiate the two Brassica species (Supplementary Fig. 29). This is in agreement with previous comparative studies based on chromosome painting12,23 and genetic mapping24,25. The extensive chromosome reshufing in Brassica is in contrast to that observed in other taxa, such as the highly syntenic tomatopotato and pearapple genomes, each with longer divergence times and less genome rearrangement26,27. This difference may be a consequence of mesopolyploidy in Brassica.

Greater TEs accumulation in B. oleracea than B. rapa. Both retro- (22.13%) and DNA (16.67%) TEs appear to be greater amplied in B. oleracea relative to B. rapa (9.43 and 12.04%) (Fig. 2a and Supplementary Table 13). We constructed 1,362 gap-free contig-contig syntenic regions by clustering orthologousB. rapaB. oleracea genes using MCscan (Supplementary Figs 29 and 30). The B. oleracea TE length (34.03% of the 259.6M) is 3.4 times greater than that of the syntenic B. rapa regions (16.73% of the 155.0M) (Fig. 2c, Supplementary Tables 21 and 22, and Supplementary Fig. 31). Phylogenetic analysis revealed thatB. oleracea has both more LTR retrotransposon (LTR-RT) families, and more members in most families than B. rapa (Fig. 2d and Supplementary Figs 12, 32 and 33). Furthermore, two new lineages of LTR-RTs, Brassica Copia Retrotransposon and Brassica Gypsy Retrotransposon, were dened in both Brassica species (Supplementary Fig. 33). Analysis of LTR insertion time revealed that B98% of B. oleracea intact LTR-RTs amplied continuously over the B4 million years (MY) since the B. oleraceaB. rapa split, whereas B68% of B. rapa intact LTR-RTs amplied rapidly within the last 1 MY, predominantly in the recent 0.2 MY (Fig. 2b and Supplementary Fig. 34). Hence, LTR-RTs expanded more in the intergenic space of euchromatic regions in B. oleracea than B. rapa. This agrees with previous observations based on comparison of BAC sequences between the A and C genomes28. As a consequence of continuous TE amplication over the last 4 MY, the genome size of B. oleracea is B30% larger than that of B. rapa

Retro-transposon DNA transposon Total

250

Length or number

200

Number of intact LTR

150

100

0 0 1 2 3 4 5 6

In Bol assembly

In Bra assembly

In Bol block

In Bra block

In Bol assembly

In Bra assembly

In Bol block

In Bra block

Age of LTRs (MYA)

Bol copia Bra copia

Bol gyspy Bra gyspy Bol all Bra all

TE size (10M bp)

TE copy number (10,000)

log (Bol/Bra)

C08

Copia

A06

A05 A07

A08 A09

A07

0.1

B. oleracea

B. rapa

Figure 2 | TE comparison analyses in B. oleracea and B. rapa. (a) TE copy number and total length in each assembly and B. oleraceaB. rapa syntenic blocks. (b) The number of intact LTR (Copia-like and Gypsy-like) birthed at different times (million years ago, MYA) in the syntenic regions ofB. oleracea and B. rapa. (c) The comparison of TE distribution and composition in B. oleraceaB. rapa syntenic blocks along B. oleracea chromosomes. We divided B. oleraceaB. rapa syntenic region into non-overlapping sliding 200 kb windows to compare TE contents. For each window, the ratio log10

(B. oleracea/B. rapa) was calculated for total syntenic block length (blue line), LTR length (purple line), gene length (yellow point), exons length (red point) and intron length (green point). If B. oleracea 4 B. rapa in absolute length of TE composition in a compared window, the dot or line is above the liney 0. The corresponding B. rapa chromosome segments along B. oleracea C08 were indicated by coloured bars. All other B. oleracea chromosomes are

showed in Supplementary Fig. 31. (d) Phylogeny of the Copia-like elements as an example of LTR-RTs of the syntenic regions in B. rapa and B. oleracea. The neighbor-joining (NJ) trees were generated based on the conserved RT domain nucleotide sequences using the Kimura two-parameter method68

in MEGA4 (ref. 69).

4 NATURE COMMUNICATIONS | 5:3930 | DOI: 10.1038/ncomms4930 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4930 ARTICLE

although the two genomes share the same ploidy and are largely collinear.

Species-specic genes and tandemly duplicated genes. While the genomes of B. oleracea and B. rapa are highly similar in terms of total gene clusters/sequences and the gene number in each cluster, there are also a large number of species-specic genes in the two species. A total of 66.5% (34,237 genes) of B. oleracea genes and 74.9% (34,324) of B. rapa genes were clustered into OrthoMCL groups (Supplementary Table 23 and Supplementary Fig. 35). We identied 9,832 B. oleracea-specic and 5,735B. rapa-specic genes, of which 77% were supported by gene expression and/or a clear Arabidopsis homologue (Supplementary Table 24). Of them, 490% of these specic genes were validated for their absence in the counterpart genomes by reciprocal mapping of raw clean reads (Supplementary Tables 25 and 26). Most Brassica-specic genes are randomly distributed along the chromosomes (Supplementary Figs 36 and 37). More than 80% of the species-specic genes were surrounded by non-specic genes (Supplementary Fig. 38), suggesting that deletion of individual genes may be the major mechanism underlying gene loss and the difference in gene numbers between B. oleracea and B. rapa.

Tandem duplication produces clusters of duplicated genes and contributes to the expansion of gene families29. We identied 1,825, 2,111 and 1,554 gene clusters containing 4,365, 5,181 and 4,170 tandemly duplicated genes in B. oleracea, B. rapa and

A. thaliana, respectively (Fig. 3a, Supplementary Tables 27 and 28 and Supplementary Fig. 39). The wide range of sequence divergence of tandem gene pairs in each species suggests that tandemgene duplication occurred continuously throughout the evolutionary history of these species, rather than in discrete bursts (Supplementary Figs 40 and 41). Their continuous and asymmetrical occurrence after species divergence resulted in 522, 697 and 815 species-specic tandem clusters in the three genomes. The frequency of tandem duplication is independent of the total gene content, suggesting that genome triplication has not inhibited its occurrence. Tandemly duplicated genes are preferentially enriched for gene ontology (GO) categories related to defence response and pathways related to secondary metabolism such as indole alkaloid biosynthesis and tropane, piperidine and pyridine alkaloid biosynthesis (Fig. 3b, Supplementary Tables 2932 and Supplementary Fig. 42). Over44.0 and 51.9% of the NBS-encoding resistance genes are tandemly duplicated in B. oleracea and B. rapa, respectively (Supplementary Table 33).

Biased loss and retention of genes after WGT/WGD. Following polyploidization, reversion of gene numbers towards diploid levels through gene loss has been widely observed in plants30. However, in Brassica this only appears to be true for collinear genes in the conserved syntenic regions, with a loss of B60%

B. oleraceaB. rapaA. thaliana

815

359 378

208 153

Number of maps

187 695

421

659

396

0 00.1

0.10.2

0.20.3

0.30.4

0.40.5

0.50.6

0.60.7

0.70.8

0.80.9

0.91.0

697

522

Tandem gene number/the total gene number in individual maps

Complex I Complex III

Cytc

Complex II Complex IV

Complex V

H+ H+ H+

B. oleraceaB. rapaA. thaliana

A. thaliana 1,554

B. rapa 2,111 B. oleracea 1,825

Cytc

H+

Cytc

Number of maps

1.10.2.2 9-8-5

Succinate

1.6.5.3 61-51-47

NAD+

UQ UQ

H+

1.3.99.1 17-12-16

1.3.5.1 10-10-12

Fumarate

1.9.3.1 5-2-6

NADH

1.6.99.3 104-88-54 1.6.99.5

49-45-24

H+

H2O

ADP

ATP

0 0 00.1

0.10.2

0.20.3

0.30.4

0.40.5

0.50.6

0.60.7

0.70.8

0.80.9

0.91.0

Cytochrome c oxidase

E/B/A

PPi

3.6.1.1 16-15-8

3.6.3.6 15-15-11

H+

H2O

No. of genes with 2 or 3 paralogs / no. of total genes in individual maps

COX10

2-2-1 COX11

3-3-1

COX15 3-1-1

COX17 2-2-2

Figure 3 | The duplicated genes derived from tandem duplication and whole-genome duplications in Brassica genomes. (a) A Venn diagram showing shared and specic tandem duplication events in A. thaliana, B. rapa and B. oleracea. (b,c) Distribution of tandem genes and WGT/WGD-derived paralogues in the KEGG pathway maps in B. oleracea (bol), B. rapa (bra) and A. thaliana (ath). For each KEGG pathway map, the proportion of the number of duplicated genes or paralogues to the total genes was calculated (x axis) and the number of maps whose tandem gene proportion fell in a rangewas shown on the y axis. (d) Oxidative phosphorylation pathway enriched by WGT-derived paralogous genes in the Brassica genomes. The gene copy number for each KO enzyme in B. oleracea, B. rapa and A. thaliana were shown (dash-connected) under the KO enzyme number.

NATURE COMMUNICATIONS | 5:3930 | DOI: 10.1038/ncomms4930 | http://www.nature.com/naturecommunications

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4930

of the predicted post-triplication gene set, nearly restoring the pre-triplication gene number. This is reected in an overall retention rate of 1.2-fold of A. thaliana orthologous genes in corresponding syntenic regions (Fig. 1c and Supplementary Table 18). In contrast, in terms of genes that have no collinear gene in A. thaliana and either Brassica species (hereafter called non-collinear genes), gene retention rates is 2.5-fold theA. thaliana gene number in B. oleracea and 1.9-fold in B. rapa, both signicantly higher than the expected rates (P value o2.2e16;

Supplementary Table 34). For these retained genes, the numbers of the genes that are common in the two Brassica species are 11,746 inB. oleracea and 10,411 in B. rapa. Most of these genes are supported by expression and/or the presence of an Arabidopsis homologue (Supplementary Table 35). More than 61% of these genes have homologues present as collinear genes and 16% also are homologous to other non-collinear genes, indicating gene movement from triplicated syntenic regions and being similar to observations in A. thaliana, where half of the genes are nonsyntenic within rosids31. This suggests that the breakdown of the triplicated syntenic relationship has not only prevented gene loss and a move towards pre-triplication gene numbers but has also maintained a higher gene density, and thus maintained WGT-derived genes for species evolution.

The presence of a large number of the retained paralogous genes in the syntenic regions led us to examine whether genes in some functional categories have preferentially been over-retained, as observed in other plants29. The results indicate that WGT-produced paralogous genes are over-retained in GO categories associated with regulation of metabolic and bio-synthetic processes, RNA metabolism and transcription factors (Supplementary Table 36 and Supplementary Figs 4345), and the two Brassica species exhibit similar patterns of gene category retention. From a study of KEGG pathways, we also found that WGT-produced Brassica paralogous genes contribute 4060% of total genes for 90% of KEGG pathways (Fig. 3c and Supplementary Fig. 43), and are functionally enriched in primary or core metabolic processes such as oxidative phosphorylation, carbon xation, photosynthesis, circadian rhythm32 and lipid metabolism (Supplementary Tables 36 and 37 and Supplementary Figs 4345). Notably, the pathways associated with energy metabolism have been enhanced in both Brassica species. For instance, in the oxidative phosphorylation pathway, there are 161 genes in A. thaliana, but 241 in B. oleracea and 208 in B. rapa. The majority (143/241 and 142/208) of these Brassica genes are multiple paralogues residing in the triplicated syntenic regions, and more than half of these paralogues have been retained as three copies, signicantly higher than observed for other genes in the triplication regions (Fig. 3d and Supplementary Fig. 43).

Phylogenetic analyses show that WGT led to an expansion of genes involved in auxin functioning (AUX, IAA, GH3, PIN, SAUR, TAA, TIR, TPL and YUCCA), morphology specication (TCP), and owering time control (FLC, CO, VRN1, LFY, AP1 and GI) (Supplementary Table 38 and Supplementary Figs 4661), and that most Arabidopsis genes in these families have two or three orthologs in Brassica species. These WGT-produced duplicated genes may provide important sources of evolutionary innovation33 and contribute to the extreme morphological diversity in Brassica species.

Divergence of duplicated genes in the Brassica genomes. The largest genetic foundation for plant genome evolution and new species formation is the differentiation of retained paralogous and orthologous genes. Around 38% (4,302/11,493) of all paralogous gene pairs in B. oleracea and B36% (4,089/11,448) in B. rapa

have different predicted exon numbers (Supplementary Data 1, Supplementary Tables 39 and 40 and Supplementary Fig. 62). There are 6,571 orthologous gene pairs with different exon numbers, accounting for 27.6% of total gene pairs (23,823). Some paralogous or orthologous pairs have high Ks values and low sequence similarity (Supplementary Fig. 63), indicating sequence differentiation. Of these paralogous genes, some offer appreciable opportunity for non-reciprocal DNA exchanges (gene conversion). About 8% of the 4,296 homologous quartets in B. rapa andB. oleracea have been affected by gene conversion (Fig. 4a, Supplementary Table 41 and Supplementary Fig. 64) and about one-sixth (53) of converted genes were inferred to have experienced independent conversion events in both Brassica species, a parallelism sometimes observed in other plants11,34. Around 4044% of conversion events involved paralogues in the less-fractionated subgenomes LF in both species, substantially higher than the other two subgenomes (Supplementary Table 41). This nding suggests that gene conversion is related to homologous gene density, which determines the likelihood of illegitimate recombination.

Analysis of RNA-seq data generated from callus, root, leaf, stem, ower and silique of B. oleracea and B. rapa suggests that 440% of WGT paralogous gene pairs are differentially expressed in these species (Fig. 4b and Supplementary Fig. 65), suggesting potential subfunctionalization of these genes. In both species, a general trend of expression differentiation was alpha-WGD paralogous genes (B46%) 4 WGT paralogous genes (B42%) 4 tandemly duplicated genes (B35%) (Fig. 4b, Supplementary Fig. 66 and Supplementary Tables 42 and 43). Different tissues harbour approximately the same number of differentially expressed duplicates, but this number was slightly higher in ower tissue. The expression level of genes in the LF subgenome was signicantly higher than corresponding syntenic genes in the more fractionated subgenomes (MF1 and MF2) while no expression dominance relationship was observed between the subgenomes MF1 and MF2 (Fig. 4c, Supplementary Table 44 and Supplementary Fig. 67). Duplicated transcription factor gene pairs showed less differentiated expression (B38%) than the expected ratio at the genome-wide level (Fig. 4d and Supplementary Table 45), while paralogues with GO categories related to membrane, catalytic activity and defence response exhibited a higher ratio of differentiated expression (Fig. 4e and Supplementary Table 46). Of B. oleraceaB. rapa orthologous gene pairs (23,823 in total), B42% were differentially expressed across all tissues (Supplementary Tables 42 and 43).

Furthermore, many paralogues generate different transcripts, resulting in expression differentiation. Analysis of AS variants of paralogous gene pairs that have identical numbers of exons demonstrated that these variants (either different variants or differential expression of the same variants) cause 420% and 444% of such paralogous genes to be differentially expressed inB. oleracea and B. rapa, respectively (Fig. 4f and Supplementary Table 47). For orthologous gene pairs of B. oleracea and B. rapa,35.5% (8,467) of gene pairs showed differential expression due to AS variation. When only counting intron retention and exon skipping, 9.3% (2,215) of gene pairs differ. Divergence in AS variants of gene pairs presents an important layer of gene regulation, as reported3538, and thus provides a genetic basis for species evolution and new species formation.

Unique GSLs metabolism pathways. GSLs and hydrolysis products have been of long-standing interest due to their role in plant defence and anticancer properties. Compared with B. rapa andB. napus, B. oleracea has the greatest GSL prole diversity, with wide qualitative and quantitative variation39,40. We identied 101

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C03

C02

Bol Bra

a - WGD

Percentage (%) of differentially expressed

duplicated gene pairs

C04

WGT

C01

C05

C06

C08

C09

C07

Tissues

MF1

Bol

Bra

** **

B. oleracea B. rapa

Regulation of transcription, DNA-dependent

C,R,St,F,Si C,R,St,F,Si

St,F,Si

C,R,St,F,Si

Sequence-specic DNA binding transcription factor activity

Total TFs pairs

C,R,St,F,Si

Log- expression value

GO total pairs

(background)

Leaf

Total pairs (background)

Flower

Percentage (%) of differentially expressed duplicated gene pairs

e f

Bol

Bra

MF2

Regulation of defense response

** St F,Si

Ratio to total gene pairs with same exon number

Ratio to total gene pairs

DNA repair

Electron carrier activity

Transmembrane transport

Flavonoid biosynthetic process

C,St,F

** C,St C,Si

Percent (%)

Anchored to plasma membrane

****

R C,L

St,L,F

Amino acid transmembrane transporter activity

R,St,F C,R

Catalytic activity

L ** C,St,F,Si

C,St,F,Si

GO total pairs

0 Bol Bra

Bol-Bra

Bol Bra

Bol-Bra

All AS

AS (IRES)

Percentage (%) of differentially expressed duplicated gene pairs

Figure 4 | Divergence of Brassica paralogous and orthologous genes in B. oleracea and B. rapa. (a) Genome-wide gene conversion in B. oleracea. The conversion in B. rapa is showed in Supplementary Fig. 64. (b) The ratio of differentially expressed duplicated gene pairs derived from different duplications: alpha whole-genome duplication (a-WGD), Brassiceae-lineage WGT, tandem duplication (TD). Bol, B. oleracea; Bra, B. rapa. C: callus; R: root;

St: stem; L: leaf; F: ower; Si: silique. The differentially expressed duplicated gene pairs were dened as fold change 42 and false discovery rate (FDR) o0.05 or gene pair where expression was detected for only one gene within gene pairs (FDR o0.05). (c) Box and whisker plots for differentiated expression for three subgenomes (LF, MF1 and MF2) in ower tissue of B. oleracea and B. rapa. For the other tissues, see Supplementary Fig. 67. (d) The duplicated gene pairs belonging to transcription factors (TFs) and its related GO terms contain a signicantly lower ratio of differentially expressed duplicated gene pairs than the average at the genome-wide level in leaf (values given) and other tissues (values not presented) (Supplementary Table 45). (e) The GO terms (left) in which the duplicated gene pairs contain a signicantly higher ratio of differentially expressed duplicated gene pairs than the average ratio at the genome-wide level in leaf and other tissues (Supplementary Table 46). Values from one tissue were presented and the other tissues were indicated with abbreviated letters to the right if expression in these tissues is signicantly higher. (f) Expression variation caused by divergence (either different variants or differential expression of the same variants) of alternative splicing (AS) variants in WGT paralogous gene pairs with identical numbers of exons and in BolBra orthologous gene pairs. IRES denotes types of intron retention and exon skipping.

and 105 GSL biosynthesis genes in B. rapa and B. oleracea, respectively, and 22 GSL catabolism genes in each species (Fig. 5a, Supplementary Table 48 and Supplementary Data 2). In the GSL biosynthesis and catabolism pathways, tandem genes (41.4%,40.7% and 33.9% in A. thaliana, B. oleracea and B. rapa, respectively) were present in a much higher proportion than the

genome-wide average (Supplementary Table 32). The observed variation of GSL proles is mainly attributed to the duplication of two genes, methylthioalkylmalate (MAM) synthase and 2-oxoglutarate-dependent dioxygenase (AOP).

In Arabidopsis, the MAM family contains three tandemly duplicated and functionally diverse members (MAM1, MAM2

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and MAM3), and functional analysis demonstrated that MAM2 (absent in ecotype Columbia) and MAM1 catalyses the condensation reaction of the rst and the rst two elongation cycles for the synthesis of dominant 3 and 4 carbon (C) side-chain aliphatic GSLs, respectively40,41, while MAM3 is assumed to contribute to the production of all GSL chain lengths42. InB. rapa and B. oleracea, MAM1/MAM2 genes experienced independent tandem duplication to produce 6 and 5 orthologs respectively (Fig. 5b,c). The main GSLs in B. oleracea are 4C and 3C GSLs (progoitrin, gluconapin, glucoraphanin and sinigrin)43, while those in B. rapa are 4C and 5C GSLs (gluconapin and glucobrassicanapin)39 (Fig. 5a). Based on the results of expression and phylogenetic analyses, we found a pair of genes Bol017070 and Bra013007, which are the only orthologous genes showing high expression in B. oleracea but silenced in B. rapa (Fig. 5a).

This expression difference most likely leads to greater accumulation of the 3C GSL anticancer precursor sinigrin inB. oleracea. Meanwhile, the expression level of MAM3 in B. rapa is much higher than in B. oleracea, explaining the accumulation of 5C GSL glucobrassicanapin in B. rapa. Other genes affecting specic anticancer GLS products are AOPs. Previously, research has reported four gene loci involved in the side-chain modications of aliphatic GSLs in Arabidopsis. Two tandemly duplicated genes AOP2 and AOP3 catalyse the formation of alkenyl and hydroxyalkyl GSLs, respectively. When both AOPs are non-functional, the plant accumulates the precursor methylsulnyl alkyl GSL. We identied three AOP2 genes inB. oleracea (Fig. 5d), but two are non-functional due to the presence of premature stop codons. In contrast, all three AOP2 copies are functional in B. rapa44. No AOP3 homologue has been

Bra029355 Bol020647 Bol020646 Bra029356 AtMAM2 AtMAM1 Bra021947 Bol037823 Bra013007 Bol017070 AtMAM3 Bra013011 Bol017071 Bra013009 Bra018524 Bol016496 AtIPMS2 AtIPMS1

Methionine

Biosynthesis

MAM

AOP Bol030626

Bol030627 Bra000847 Bra034181 Bra034182 AtAOP1 Bra000848 Bra018992 AtAOP3

AtAOP2 BolAOP2 BolAOP2-m1 Bra018521 BolAOP2-m2 Bra034180

BCAT4 [1, 2, 2]

MAM1/2 [2, 6, 5]

MAM3 [1, 1, 1]

CYP79F1 [1, 1, 1] CYP79F2 [1, 0, 0]

CYP83A1 [1, 2, 2]

GGP1 [1, 3, 3] SUR1 [1, 2, 4]

UGT74B1 [1, 1, 1] UGT74C1 [1, 2, 2]

ST5b [1, 2, 2] ST5c [1, 1, 1]

100

2-keto acid

Chain elongated methionine

Aldoxime

aci-nitro compound

S-alkyl-thiohydroximate

Thiohydroximate

Desulpho-GSL

Indole-3-acetaldoxime

S-indol-thiohydroximate

Thiohydroximate

Desulpho-GSL

100

Typtophan

CYP79B2 [1, 3, 3] CYP79B3 [1, 1, 1]

CYP83B1 [1, 2, 1]

GGP1 [1, 3, 3] SUR1 [1, 2, 4]

UGT74B1 [1, 1, 1]

ST5a [1, 2, 2]

GSTF11 [1, 1, 1] GSTF20 [1, 1, 1]

100

1-aci-nitro-2-indolyl-ethane

100

ST5b [1, 2, 2] ST5c [1, 1, 1]

4MTB (glucoerucin)

I3M (glucobrassicin)

CYP81F4 [1, 2, 3]

GSTF9 [1, 2, 4] GSTF10 [1, 1, 1]

CYP81F1 [1, 2, 4] CYP81F2 [1, 3, 3] CYP81F3 [1, 1, 1]

4MSOB(glucoraphanin) 3MSOP

(glucoiberin)

3PREY (sinigrin)

100

IGMT1 [1, 3, 2] IGMT2 [1, 2, 1]

PEN2 [1, 2, 1]

1OHI3M

1MOI3M

4OHI3M

4MOI3M

AOP2 [1, 3, 3 (2)]

GS-OH [1, 3, 2]

AOP2 [1,3,3 (2)]

ESP [1, 2, 5] TFP

IGMT1 [1, 3, 2] IGMT2 [1, 2, 1]

4BTEY (gluconapin)

4OHB (progoitrin)

3MTP (glucoibervirin)

Aglucone

Nitrile

Aliphatic GSLs Indolic GSLs

1,000

2,000 bp

Breakdown

TGGs [4, 6, 4]

TFP

BolAOP2

ESP [1, 2, 5] NSPs [2, 3, 2] TFP

Thiocyanate

Isothiocyanate sulphoraphane, iberin, erucin, AITC, I3C, DIM

BolAOP2-m1

BolAOP2-m2

Epithionitrile

Figure 5 | Whole-genome-wide comparison of genes involved in glucosinolate metabolism pathways in B. oleracea and its relatives. (a) Aliphatic and indolic GSL biosynthesis and catabolism pathways in A. thaliana, B. oleracea and B. rapa. The copy number of GSL biosynthetic genes in A. thaliana,B. rapa and B. oleracea are listed in square brackets, respectively. Potential anticancer substances/precursors are highlighted in blue bold. Two important amino acid chain elongation and side-chain modication loci MAMs and AOP2 are highlighted in red bold, within the number in the green bracket representing the number of non-functional genes. (b,c) The neighbour-joining (NJ) trees of MAM and AOP genes were generated based on the aligned coding sequences and 100 bootstrap repeats. The silenced genes are indicated by red hollow circle, expressed functional genes are represented by red solid disc and green rectangle. In A. thaliana ecotype Columbia there are just MAM1 and MAM3. (d) Three B. oleracea AOP2 loci among which are one functional AOP2 and two mutated AOP2. 1MOI3M: 1-methoxyindol-3-ylmethyl GSL; 1OHI3M: 1-hydroxyindol-3-ylmethyl GSL; 3MSOP: 3-methylsulnylpropylGSL; 3MTP: 3-methylthiopropyl GSL; 3PREY: 2-Propenyl GSL; 4BTEY: 3-butenyl GSL; 4MOI3M: 4-methoxyindol-3-ylmethyl GSL; 4OHB, 4-hydroxybutyl GSL; 4OHI3M: 4-hydroxyindol-3-ylmethyl GSL; 4MSOB: 4-methylsulnylbutyl GSL; 4MTB, 4-methylthiobutyl GSL; AITC: allyl isothiocyanate;

I3C: indole-3-carbinol; I3M: indolyl-3-methyl GSL; DIM: 3,30-diindolymethane; MAM: methylthioalkylmalate; AOP: 2-oxoglutarate-dependent dioxygenase.

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identied in Brassica. This analysis supports GSL content surveys and explains why glucoraphanin is abundant in B. oleracea, but not in B. rapa.

DiscussionThe Brassica genomes experienced WGT11,12,25 followed by massive gene loss and frequent reshufing of triplicated genomic blocks. Analysis of retained or lost genes following triplication identied over-retention of genes for metabolic pathways such as oxidative phosphorylation, carbon xation, photosynthesis and circadian rhythm32, which may contribute to polyploid vigour45. Fewer lost genes were observed in the less-fractionated subgenome, possibly due to expression dominance as reported in maize46.

Gene expression analysis revealed extensive divergence and AS variants between duplicate genes. This subfunctionalization or neofunctionalization of duplicated genes provides genetic novelty and a basis for species evolution and new species formation. For example, TF genes that are considered to be conserved still have more than 38% of paralogous pairs showing differential expression across tissues although this percentage is lower than the average from all duplicated genes. Gene expression variation may contribute to an increased complexity of regulatory networks after polyploidization.

The multi-layered asymmetrical evolution of the Brassica genomes revealed in this study suggests mechanisms of polyploid genome evolution underlying speciation. Asymmetrical gene loss between the Brassica subgenomes, the asymmetrical amplication of TEs and tandem duplications, preferential enrichment of genes for certain pathways or functional categories, and divergence in DNA sequence and expression, including alternative splicing among a large number of paralogous and orthologous genes, together shape a route for genome evolution after polyploidization. A molecular model of polyploid genome evolution through these asymmetrical mechanisms is summarized in Supplementary Fig. 2. The additional information of accessible large datasets and resource was provided in Supplementary Table 49.

In summary, the B. oleracea genomic sequence, its features in comparison with its relatives, and the genome evolution mechanisms revealed, provide a fundamental resource for the genetic improvement of important traits, including components of GSLs for anticancer pharmaceuticals. The genome sequence has also laid a foundation for investigation of the tremendous range of morphological variation in B. oleracea as well as supporting genome analysis of the important allotetraploid cropB. napus (canola or rapeseed).

Methods

Sample preparation and genome sequencing. A B. oleracea sp. capitata homozygous line 0212 with elite agronomic characters and widely used as a parent in hybrid breeding was used for the reference genome sequencing (Supplementary Methods). The seedlings of plants were collected and genomic DNA was extracted from leaves with a standard CTAB extraction method. Illumina Genome Analyser whole-genome shotgun sequencing combined with GS FLX Titanium sequencing technology was used to achieve a B. oleracea draft genome. We constructed a total of 35 paired-end sequencing libraries with insertion sizes of 180 base pairs (bp), 200 bp, 350 bp, 500 bp, 650 bp, 800 bp, 2 kb, 5 kb, 10 kb and 20 kb following a standard protocol provided by Illumina (Supplementary Methods). Sequencing was performed using Illumina Genome Analyser II according to the manufacturers standard protocol.

Genome assembly and validation. We took a series of checking and ltering measures on reads following the Illumina-Pipeline, and low-quality reads, adaptor sequences and duplicates were removed (Supplementary Methods). The reads after the above ltering and correction steps were used to perform assembly including contig construction, scaffold construction and gap lling using SOAPdenovo1.04 (http://soap.genomics.org.cn/

Web End =http://soap.genomics.org.cn/) (Supplementary Methods). Finally, we used 20-kb-span paired-end data generated from the 454 platform and 105-kb-span BAC-end

data downloaded from NCBI (http://www.ncbi.nlm.nih.gov/nucgss?term=BOT01

Web End =http://www.ncbi.nlm.nih.gov/nucgss?term=BOT01) to extend scaffold length (Supplementary Methods). The B. oleracea genome size was estimated using the distribution curve of 17-mer frequency (Supplementary Methods).

To anchor the assembled scaffolds onto pseudo-chromosomes, we developed a genetic map using a double haploid population with 165 lines derived from a F1 cross between two homozygous lines 0212 (sequenced) and 0188 (re-sequenced). The genetic map contains 1,227 simple sequence repeat markers and single nucleotide polymorphism markers in nine linkage groups, which span a total of 1,180.2 cM with an average of 0.96 cM between the adjacent loci16. To position these markers to the scaffolds, marker primers were compared with the scaffold sequences using e-PCR (parameters -n2 -g1 d 400800), with the best-scoring match chosen in case of multiple matches.

We validated the B. oleracea genome assembly by comparing it with the published physical map constructed using 73,728 BAC clones (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/

Web End =http:// http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/

Web End =lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/ )17 and a genetic map fromB. napus18 (Supplementary Methods). Eleven Sanger-sequenced B. oleracea BAC sequences were used to assess the assembled genome using MUMmer-3.22 (http://mummer.sourceforge.net/

Web End =http://mummer.sourceforge.net/) (Supplementary Methods).

Gene prediction and annotation. Gene prediction was performed on the genome sequence after pre-masking for TEs (Supplementary Methods). Gene prediction was processed with the following steps: (i) De novo gene prediction used AUGUSTUS47 and GlimmerHMM48 with parameters trained from A. thaliana genes. (ii) For homologue prediction, we mapped the protein sequences fromA. thaliana, O. sativa, C. papaya, V. vinifera and P. trichocarpa to the B. oleracea genome using tblastn with an E-value cutoff of 10 5, and used GeneWise (Version2.2.0)49 for gene annotation. (iii) For EST-aided annotation, the Brassica ESTs from NCBI were aligned to the B. oleracea genome using BLAT (identity Z0.95, coverage Z0.90) and further assembled using PASA50. Finally, all the predictions were combined using GLEAN51 to produce the consensus gene sets.

Functional annotation of B. oleracea genes was based on comparison with SwissProt, TrEMBL, Interproscan and KEGG proteins databases. The tRNA genes were identied by tRNAscan-SE using default parameters52. Then rRNAs were compared with the genome using blastn. Other non-coding RNAs, including miRNA, snRNA, were identied using INFERNAL53 by comparison with the Rfam database.

TE annotation. LTR-RTs were initially identied using the LTR_STRUC54 programme, and then manually annotated and checked based on structure characteristics and sequence homology. Rened intact elements were then used to identify other intact elements and solo LTRs55. All the LTR-RTs with clear boundaries and insertion sites were classied into superfamilies (Copia-like, Gypsy-like and Unclassied retroelements) and families relying on the internal protein sequence, 50, 30 LTRs, primer-binding site and polypurine tracts. Non-LTR-RTs (Long interspersed nuclear element, LINE and Short interspersed nuclear element, SINE) and DNA transposons (Tc1-Mariner, hAT, Mutator, Pong, PIF-Harbinger, CACTA and miniature inverted repeat TE) were identied using conserved protein domains of reverse transposase or transposase as queries to search against the assembled genome using tblastn. Further upstream and downstream sequences of the candidate matches were compared with each other to dene their boundaries and structure56. Helitron elements were identied by the HelSearch 1.0 programme57 and manually inspected. All the TE categories were identied according to the criteria described previously58. Typical elements of each category were selected and mixed together as a database for RepeatMasker59 analysis. Around 20 coverage of shotgun reads randomly sampled from the two Brassica

genomes were masked by the same TE data set to conrm the different accumulation of TEs between the two genomes.

Syntenic block construction of B. oleracea and its relatives. We used the same strategy as described in the B. rapa genome paper11 to construct syntenic blocks between species (Supplementary Methods). The all-against-all blastp comparison (E-value r 1e5) provided the gene pairs for syntenic clustering determined by

MCScan (MATCH_SCORE: 50, MATCH_SIZE: 5, GAP_SCORE: 3, E_VALUE: 1E05). As applied in B. rapa11, we assigned and partitioned multiple B. oleracea orB. rapa chromosomal segments that matched the same A. thaliana segment (A to X numbering system in A. thaliana22) into three subgenomes: LF, MF1 and MF2.

OrthoMCL clustering. To identify and estimate the number of potential orthologous gene families between B. oleracea, B. rapa, A. thaliana, C. papaya,P. trichocarpa, V. vinifera, S. bicolor and O. sativa, and also between B. oleracea andB. rapa, we applied the OrthoMCL pipeline60 using standard settings (blastp E value o1 10 5 and ination factor 1.5) to compute the all-against-all

similarities.

Phylogenetic analysis of gene families. We performed comparative analysis of trait-related gene families. Genes from grape, papaya and Arabidopsis were downloaded from the GenoScope database (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/

Web End =http://www.genoscope.cns.fr/externe/

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http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/

Web End =GenomeBrowser/Vitis/ ), the Hawaii Papaya Genome Project (http://asgpb.mhpcc.hawaii.edu/papaya/

Web End =http://asgpb.mhpc http://asgpb.mhpcc.hawaii.edu/papaya/

Web End =c.hawaii.edu/papaya/ ), and the Arabidopsis Information Resource (http://www.arabidopsis.org/

Web End =http:// http://www.arabidopsis.org/

Web End =www.arabidopsis.org/ ). Previously reported Arabidopsis and Brassica gene sequences were downloaded from TAIR (http://www.arabidopsis.org/

Web End =http://www.arabidopsis.org/) and BRAD (http://brassicadb.org/brad/

Web End =http://brassicadb.org/brad/). The protein sequences of the genes were used to determine homologues in grape, papaya, Arabidopsis, B. oleracea and B. rapa by performing blast comparisons with an E-value 1e10. The Clustal61 programs were used for multiple sequence alignment. Alignment of the small family of GI genes was performed using MEGA562 to conduct neighbour-joining analysis with default parameters and subjected to careful manual checks to remove highly divergent sequences from further analysis. While for other genes, often found in families of tens of genes, the phylogenetic analysis were performed by PhyML63, which can accommodate quite divergent sequences by implementing a maximal likelihood approach with initial analysis based on neighbour-joining method. During these analyses, we constructed trees using both CDS and protein sequence, and the protein-derived tree was used to show the phylogeny if not much incongruity was found. Bootstrapping was performed using 100 repetitive samplings for each gene family. All the inferred trees were displayed using MEGA5 (ref. 62). The multiple sequence alignment of these families was provided as Supplementary Data 3.

Differential expression of duplicated genes across tissues. RNA-seq reads were mapped to their respective locations on the reference genome using Tophat64. Uniquely aligned read counts were calculated for each gene for each tissue sample. We performed the exact conditional test of two Poisson rates on read counts of duplicated genes to test the differential expression of duplicated genes, according to the method applied in soybean65,66. For each duplicated gene pair (for example, genes A and B), read counts and gene length were denoted as Ea and La for gene A, and Eb and Lb for gene B, respectively. The read counts of the genes A and B were assumed to follow the Poisson distributions with rates lA Ra La and

lB Rb Lb. Under the null hypothesis of equal expression of the genes A and B,

that is, Ra Rb, the conditional distribution of Ea given Ea Eb k follows a

binomial distribution with success probability P la/(la lb) La/(La Lb).

The P values were computed and further adjusted to maintain the false discovery rate at 0.05 across gene pairs using the BenjaminiHochberg method67.

Statistical analysis. The average number of all retained orthologues in the three subgenomes was used to estimate the expected retained gene number in each block, and used together with the observed retained gene number, for the gene retention disparity statistics using the w2 test. In the GO, IPR (Interproscan) or KEGG enrichment analyses of WGT or tandem genes, the w2 test (N45) or the Fishers exact test (Nr5) was used to detect signicant differences between the proportion of (WGT or tandem) genes observed in each child GO, IPR or KEGG categories, and the expected overall proportion of (WGT or tandem) genes in the whole genome. Correlation of the gene numbers of WGT-derived paralogous genes with tandem genes in 938 GO terms was tested by Pearson correlation coefcients (Supplementary Figure 68). The BenjaminiHochberg false discovery rate was performed to adjust the P values67.

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Acknowledgements

This work was supported by the National Basic Research Program of China (2011CB109300, 2012CB113906, 2012CB723007 and 2006CB101600), the National Natural Science Foundation of China (3067134, 30671119 and 31301039), the National High Technology Research and Development Program (2013AA102602, 2012AA100105 and 2012AA100104), the China Agriculture Research System (CARS-13 and CARS-25-A), the Core Research Budget of the Non-prot Governmental Research Institution (1610172010005), the Special Fund for Agro-scientic Research in the Public Interest (201103016), ChinaAustralia collaboration project (2010DFA31730), UK Biotechnology and Biological Sciences Research Council (BB/E017363/1), the Australian Research Council (LP0882095, LP0883462, DP0985953 and LP110100200), the Next-Generation BioGreen 21 Program (PJ008944 and PJ008202), and the US National Science Foundation (IOS 0638418, DBI 0849896, MCB 1021718).

Author contributions

I.B., B.C., D.E., Q.H., W.H., G.J.K., S.L., Y.L., J. Ma, A.H.P., J.C.P., I.A.P.P., JunW., XiaowuW., XiyinW. and T.-J.Y. are principal investigators (alphabetic order). B.C., W.H., A.H.P., JunW. and XiaowuW. are equally contributing senior authors. S.L., J.W., W.H.,X.X. and Z.Y. planned and managed the project. S.L., C.T., A.H.P. and D.E., X.Y. and M.Z. wrote this manuscript and I.B., J. Ma., G.J.K., J.C.P., B.C., T.-J.Y., I.A.P.P., XiyinW., XiaowuW., K.L., Y.L., J.B. and A.G.S. made revision or edits or comments. J.W. (leader), W.H. (co-leader), JunW., L.Y., and Z.Y. performed DNA sequencing. L.Y. (leader), W.H. (co-leader), S.H., J.W., S.L. and J.Y. conducted genomic sequence assembly. S.H. (leader), XiyinW. (co-leader), J.Min, I.B., W.H., J.B., D.E., P.R., S.L., J.S., Y.L. and W.W. conducted scaffold anchoring to linkage maps and assembly validation. X.Y. (leader), J.Y. (co-leader), S.L., Q.Z., S.H. and J. Min performed annotation. C.T. (leader), Wanshun L., W.H., Y.L., C.L., W.W., J. Wu, S.L., C.D. and M.Z. performed transcriptome sequencing. S.L. conceived analysis of comparison and evolution. S.L. (leader), C.T., X.Y., ZhangyanW., C.L., S.H., J. Ma, J.Y., M.Z., Zhuo W., Q.Z., S.P., I.A.P.P., A.G.S., L.Y., I.B., G.J.K., J.C.P., XiaowuW., B.C., F.C., YinH., WenbinL. and X.Liang performed analysis of comparative genomics and evolution. J. Ma (leader), M.Z., Q.Z., C.T., S.L., B.C., S.H., H.B., C.L. and JianaL. conducted TE analysis. XiyinW. (leader), J.Y., T.-J.Y., ZhangyanW., L.W., J. Li, T.-H.L., JinpengW., H.J., X.T., X.L., M.G. and L.J. conducted gene family analysis. K.L. (leader), J.Y., S.L., C.T., H.L., H.G., S.P., D.Z., Z.F., Q.H., Xnfa W., C.Q., D.D., Z.H., Y.H., J.H., D.M., J.L., Z. Li, J.Z., L.X., Y.Zhou., Z.L. and Y.Zhang conducted trait-related gene analysis. A.H.P. (leader), XiyinW., D.J., Y.W. and T.-H.L. conducted gene conversion analysis. T.-J. Y. (leader), M.Z., P.S., B.-S.P., J.Ma, N.E.W., R.Q., X.L., J.Lee and H.H.K. conducted centromere analysis. C.T. (leader), S.L., X.Y., S.H., C.L., Zhangyan W., Q.Z., J.Y., J.T. and J.B. conducted tandemly duplicated gene analysis. ZhangyanW. and J.Y. performed data submission.

Additional information

Accession codes: Genome sequence data for B. oleracea have been deposited in the DDBJ/EMBL/GenBank nucleotide core database under the accession code AOIX00000000. Transcriptome sequence data for B. rapa and B. oleracea have been deposited in the DDBJ/EMBL/GenBank Sequence Read Archive (SRA) under the accession codes GSE43245 and GSE42891 respectively.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Liu, S. et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 5:3930 doi: 10.1038/ncomms4930 (2014).

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NATURE COMMUNICATIONS | 5:3930 | DOI: 10.1038/ncomms4930 | http://www.nature.com/naturecommunications

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Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

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Title

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

Author

Liu, Shengyi; Liu, Yumei; Yang, Xinhua; Tong, Chaobo; Edwards, David; Parkin, Isobel A P; Zhao, Meixia; Ma, Jianxin; Yu, Jingyin; Huang, Shunmou; Wang, Xiyin; Wang, Junyi; Lu, Kun; Fang, Zhiyuan; Bancroft, Ian; Yang, Tae-jin; Hu, Qiong; Wang, Xinfa; Yue, Zhen; Li, Haojie; Yang, Linfeng; Wu, Jian; Zhou, Qing; Wang, Wanxin; King, Graham J; Pires, J Chris; Lu, Changxin; Wu, Zhangyan; Sampath, Perumal; Wang, Zhuo; Guo, Hui; Pan, Shengkai; Yang, Limei; Min, Jiumeng; Zhang, Dong; Jin, Dianchuan; Li, Wanshun; Belcram, Harry; Tu, Jinxing; Guan, Mei; Qi, Cunkou; Du, Dezhi; Li, Jiana; Jiang, Liangcai; Batley, Jacqueline; Sharpe, Andrew G; Park, Beom-seok; Ruperao, Pradeep; Cheng, Feng; Waminal, Nomar Espinosa; Huang, Yin; Dong, Caihua; Wang, Li; Li, Jingping; Hu, Zhiyong; Zhuang, Mu; Huang, Yi; Huang, Junyan; Shi, Jiaqin; Mei, Desheng; Liu, Jing; Lee, Tae-ho; Wang, Jinpeng; Jin, Huizhe; Li, Zaiyun; Li, Xun; Zhang, Jiefu; Xiao, Lu; Zhou, Yongming; Liu, Zhongsong; Liu, Xuequn; Qin, Rui; Tang, Xu; Liu, Wenbin; Wang, Yupeng; Zhang, Yangyong; Lee, Jonghoon; Kim, Hyun Hee; Denoeud, France; Xu, Xun; Liang, Xinming; Hua, Wei; Wang, Xiaowu; Wang, Jun; Chalhoub, Boulos; Paterson, Andrew H

Pages

3930

Publication year

2014

Publication date

May 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms4930

ProQuest document ID

1527392805

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

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